Alkaline phosphatase activity in the supernatant from three different co‐culture systems was measured on day 7 after challenged with/without Ti particles. In preparation for this assay, medium was collected and centrifuged to remove cell debris and Ti particles. ALP activity was assayed using an Alkaline Phosphatase Assay Kit (Sigma‐Aldrich). In brief, MC3T3‐E1 cells were collected using cell scrapers not trypsin. Treated with lysis buffer (without PMSF), and put on ice for 20 min, then centrifuged at 21,130 g for 15 min, collected the supernatant and mixed the centrifuged medium. The substrate solution pNPP prepared according to the instructions was added and incubated the 96‐well plates in the dark for 30 min at room temperature. After NaOH terminated the reaction, the absorbance was read at 405 nm.
Three different co‐culture models were maintained as described above. ALP staining was performed on day 7 after challenge with Ti particles. MC3T3‐E1 cells were stained using an Alkaline Phosphatase Stain Kit (Jiancheng). In brief, cells were fixed in methanol and added with 5‐bromo‐4‐chloro‐3‐indolyl phosphate plus nitroblue tetrazolium chloride in Tris–HCl, NaOH and MgCl2 successively, then incubated at room temperature for 2 h in the dark.
Three different co‐culture models were maintained as described above. ALP staining was performed on day 7 after challenge with Ti particles. MC3T3‐E1 cells were stained using an Alkaline Phosphatase Stain Kit (Jiancheng). In brief, cells were fixed in methanol and added with 5‐bromo‐4‐chloro‐3‐indolyl phosphate plus nitroblue tetrazolium chloride in Tris–HCl, NaOH and MgCl2 successively, then incubated at room temperature for 2 h in the dark.